Susan Taylor (UCSD) Part 1: Protein Phosphorylation in Biology

My name is Susan Taylor. I’m a professor of chemistry and biochemistry and also of pharmacology at the University of California, San Diego. I’m also an investigator of the Howard Hughes Medical Institute. And what I’d like to do today is to go through three lectures and hopefully leave you with three general concepts. First, I’d like to point out why protein phosphorylation is so important in biology. Then, in the second lecture, I’d like to introduce you to the protein kinase molecular and how that functions. And in the third lecture, I’d like to focus on how protein kinases are regulated and localized. So if we go to the central dogma of biology, where DNA makes RNA makes proteins This is quite an extraordinary era that we live in now; this genomic era of science where we have so much information available to us. So if we look at DNA: DNA is a linear template of four bases. And the speed with which we can sequence DNA is just beyond anyone’s comprehension, even a decade ago, that we could have whole genomes so rapidly. So it’s a linear template and the information is in this linear template. And the same is true for RNA. It’s linear template of four bases and the information is encoded in that linear template. But if we look at proteins, proteins are a little bit different. They’re made up of 20 different amino acids and the chemical properties of those amino acids are quite different. And in addition, they get modified once they’re made. And that modification can make a critical difference in how they work. So, understanding how a protein works is more complicated. So with DNA and RNA, transcription transcribes the DNA into RNA. They’re both linear templates. If you look at proteins and translation now: RNA is translated into proteins, where you have all these diverse amino acids and if we look at a protein’s sequence, it, of course, defines the chemical composition of that structure of that protein molecule. But it doesn’t really tell you how it works. And to really understand how a protein works, you need to have structure so that you know where those amino acids are, you know how they work together to create an active and functional protein. And understanding this is much more complicated than just reading out the template from the DNA or the RNA. And so I like to think of our present era of science not as the genomic era of science but the proteo-genomic era of science. And ultimately we’re going to have to understand this erite gamut going from DNA to RNA to proteins. And it’s going to be much more challenging to do the proteins but we’re already making enormous progress there. So what are the building blocks, the atoms, that make up proteins? So we look at carbons, nitrogens, oxygens, hydrogens. OK, those are all there. There’s also a little bit of sulfur there. But then, what about phosphates? So where do phosphates come in and why are they important? So this is the phosphate. It’s 80 kiloDaltons. It’s a little, small moiety that you add onto a very very large protein. And I like to go back to a review that Frank Westheimer did back in 1988. So he was one of the chemists who studied phosphoryl-transfer, one of the major pioneers in this area. And he elucidated the importance of phosphates for biology. So he made two major points. One is, its importance for DNA and RNA, for genetic information. and for transfer of information. And each of those linear templates for DNA and RNA are linked by phosphates. So clearly, all of this template is critically dependent on phosphate. And the other thing he recognized was the importance of phosphate for energy. And so in this case we need to go to another molecule. And this is an organelle, a mitochondria which is the powerhouse of the cell. And what the mitochondria does is to make ATP. And what ATP does is to drive all of the biological processes that take place in every one of our cells. So here’s ATP and it’s the gamma phosphate at the end that turns over and provides our cells with energy. And just to emphasize how important this is, the average 70 kilogram person turns over 40 kilograms of ATP a day. So, I always find this number astounding. So this is critically important, that phosphate for energy in our cells. What Westheimer did not address at all, and this field was just beginning to really emerge in terms of its huge importance, at that time, protein phosphorylation as a mechanism for regulating biology. And that’s what I want to try and focus on now. And we have to go back, again, to some of the history and in this arena it was Ed Krebs and Eddie Fischer who were the first to demonstrate, in the late 1950s, that phosphorylation was important for regulation of proteins. And they received the Nobel Prize for that in 1992. So, they were looking at glycogen metabolism in the liver. And this is a liver cell. The dense particles are the granules. There are also mitochondria there; you need a lot of energy for anything that a cell does. There are a lot of mitochondria. And if we look at the glycogen particles, what all of us do when we have a carbohydrate rich meal, the liver takes up that glucose and you make glycogen. You store it there. And even after a short fast, like sleeping overnight, when you wake up in the morning, you have mobilized some of that glycogen into your bloodstream so that your brain and the rest of your body still gets glucose. So you make and break down glycogen as a fundamental part of metabolism. And the enzyme that does that is called glycogen phosphorylase. It breaks down glycogen into glucose. And that’s the enzyme that Krebs and Fischer worked on. And what they discovered was this enzyme–now we know its structure. It’s very large. Each chain (it’s a dimer, there are two subunits) each has over 800 amino acids. And what they found is, if you…this exists in two different states. It can be phosphorylated, one phosphate on each chain. It can be phosphorylated or not phosphorylated. And when it is phosphorylated, it is turned on. It is an active enzyme. And when it is not phosphorylated, it is not active. And so this fundamental concept is really the essence of the importance of protein phosphorylation for regulation. So, how does it get added? How does that phosphate get added? It gets added by a protein kinase. So protein kinase uses ATP, transfers that gamma phosphate to a protein. So now you have many proteins, more than half the proteins in our bodies exist either as a dephosphorylated molecule or as a phosphorylated molecule. So they can be turned on and turned off. And the phosphatases are enzymes that take the phosphate off. So phosphates are going on and off of your proteins all the time. They’re switches, they’re molecular switches that either give a go signal or stop signal. They are essential molecular switches for all of biology. And I like to give just one example that is one of the most dynamic events that a cell does, it is to go through cell division. And this is a lily cell dividing. And you can see as this lily cell goes through the different steps of mitosis, how dynamic this is; organizing these chromosomes, then having the cell actually divide. This process is mediated, primarily, by kinases and phosphatases that get turned on and turned off and that allow mitosis to start, this phase to end, start the next phase. It’s critically regulated by kinases and phosphatases. No cell could divide without that critical, highly correlated regulation. So, let’s go back to the history. So, phosphorylase kinase is the kinase that phosphorylated glycogen phosphorylase and then the second one to be discovered is called PKA or cyclic AMP dependent protein kinase. And I’m going to tell you about those two and show you how, in this case, they work together as a team to regulate this biological event. So here’s glycogen phosphorylase when you’ve just had a carbohydrate rich meal; glucose is high, insulin is high, glucagon is low. Insulin and glucagon are two metabolic hormones that tell the body “are we in an energy rich stage, with glucose, or are we in more of a fasting state.” So it’s turned off. Then you look at glycogen phosphorylase, it’s turned off. You have lots of glucose. You want to be making glycogen, not breaking it down. OK, now you look at when glucose levels are low. You have high glucagon, low insulin. In this case you want to mobilize that glycogen that is stored in the liver and then this enzyme is turned on. And it’s turned on by the addition of that one phosphate to each of the chains that’s in the glycogen phosphorylase dimer. OK, so let’s see how that works. So here’s glucagon. Glucagon is a hormone. It doesn’t ever get into the cell. It binds to a receptor on the surface of the liver cell. And in this case, this is a GPCR (G protein coupled receptor), the largest gene family in our human genome. It binds, that couples to a heterotrimeric G protein, which becomes activated and that in turn leads to the activation of adenylate cyclase which makes cyclic AMP. So this concept is: cyclic AMP is second messenger. It allows some extracellular signal to be translated into a biological response. This was discovered by Earl Southerland earlier in the 1950s, this second messenger concept for cyclic AMP. It is conserved as a second messenger in all of biology even in bacteria. So let’s see what…this is summarizing what I just told you. Your extracellular signal, in this case glucagon, a hormone from the pancreas, binds the glucagon receptor, activates the G alpha subunit, that activates adenylate cyclase and that makes cyclic AMP. OK, what does cyclic AMP do? OK, so let’s look now at this biological response. So, here we go to PKA. And PKA, like most protein kinases, I told you they’re switches, is kept in an off state here and in this case it’s got regulatory R subunits and catalytic subunits, C subunits and when they’re together and there’s no cyclic AMP around, it is inactive, it’s turned off. And cyclic AMP…this is the main target for cyclic AMP: these regulatory subunits of PKA. It binds with very high affinity to the regulatory subunit and that then unleashes the catalytic activity. And depending on the cell type, there are many things it can do. PKA has many substrates. It regulates many aspects of biology. It can also go into the nucleus and turn on gene transcription. So, turning on one kinase can have many consequences. We’re going to focus here on this liver cell and what are the consequences for glycogen metabolism. So let’s look at this cyclic AMP. It gets made in response to glucagon. It binds to PKA and it converts it from an inactive state to an active state. OK, what does that do now with respect to glycogen metabolism? Well, glycogen phosphorylase kinase that was the first kinase that Krebs and Fischer characterized. That’s the kinase that phosphorylates glycogen phosphorylase. And PKA turns it from an off state to an on state. So we now have one kinase, PKA, turning on another kinase, glycogen phosphorylase kinase. And then, that in turn acts on glycogen phosphorylase and again, that’s converting it from an inactive state to an active state. So these on-off switches are happening all the time in our cells. So…and in these cases it’s just one phosphate. One single phosphate can make an enormous difference for a very large protein whether it’s active or whether it’s inactive. So let’s go back to the history now and look at this curve a little bit more. So in the 1980s this really expanded exponentially. And that’s because we developed the technology to clone and to sequence DNA. So from that it became clear that there were many kinases and that their sequences were all related. And we now fast-forward to the genomic era of today and we have whole kinomes from organisms. And the human kinome is about 2% of the human genome codes from protein kinases. It’s one of the largest gene families. PKA belongs to this little branch down here. And the other one that I told you about is phosphorylase kinase. It belongs to this other branch. Those are both very important, classical, metabolically important kinases. So, let’s go back to this now and look at another event that was really important around 1980. And this is the discovery that Src was also a protein kinase. And let me tell you about Src. So the history of Src: it was first discovered as an oncogene in chickens from Rous Sarcoma Virus. So the Rous Sarcoma Virus causes cancer in chickens. And so Src is responsible for that transformation of a normal chicken cell into malignant cancer cell and Src was the oncogene that was responsible for that. So that was discovered back in the 70s. 1978, it was shown that this Src also had kinase activity, protein kinase activity. And then Src was cloned. So then you had the sequence of Src. And then Tony Hunter and Bart Stefton showed that phosphotyrosine was also an important biological site for phosphorylation that we have. Coming back to here, these are all serine thronine kinases. And now we have this whole tree of tyrosine kinases. And they are related by sequence. They all belong to the same family. If we look at serine and threonine, most of those kinases on the yellow line are serine threonine kinases. They phosphorylate serine threonine and they’re much more abundant. But then you have tyrosine as another amino acid that can be phosphorylated and tyrosine is very, very important. Although not as abundant, critically important for biology and for disease. So we look at the kinome now and it’s this branch at the top that corresponds to those tyrosine kinases and all the rest of these are phosphorylating serine and threonine. So, we have a branch, a very large kinome that includes both serine and threonine kinases and tyrosine kinases. And so I want, at the end here, to tell you the importance again of adding one phosphate. As I showed for glycogen phosphorylase, one phosphate makes a difference between it being active and inactive. So, let me tell you, just for kinases in general, they not only add phosphates to other proteins, they are typically phospho-proteins themselves. And when you just encode that protein, translate that protein from the sequence, that has all the amino acids there but that kinase is not active. And typically you add one phosphate to what we call the activation loop and that converts an inactive kinase into active kinase. So, kinases themselves are highly regulated by phosphorylation. OK, so again, one phosphate. So now let’s look at Src and PKA and I’ll get more into these domains and things in the next lecture, but to point out that PKA has this kinase core which is important for its kinase activity. And Src has…its sequence is related and that conserved kinase core is what builds that whole kinome tree. All of those kinases have this core. What was unusual about Src was that it had these other domains that turned out also to be conserved sequences but they were not conserved in other kinases. A small subgroup had these domains. And these were discovered by Tony Pawson in the 1980s and were named…he named them SH3, SH2 and SH1. SH1 was the kinase domain. And then, what he found was that the SH2 domains, in particular, bind to phosphotyrosine. And so this introduced the whole concept of adaptor molecules. That this is a domain whose main function is to bind to phosphortyrosine. So it’s an adaptor. Now, let me show you how this works for Src. And Src is just an example of all of the tyrosine kinases there are. Each has a different variation on this theme. So Src…ordinarily it’s turned off. I’ve told you kinases are switches, you turn them off and you turn them on. And in this case it’s turned off by a phosphate that’s at the C-terminus or Src. And it binds to its own SH2 domain. So, it’s kind of its introverted mode. It’s not interacting with other proteins. It’s interacting with itself and turned off. And so the key event for activating Src is to remove that inhibitory phosphate. So you take it off. You take the phosphate off and then you now convert it into an active enzyme. And the first thing it does is to phosphorylate itself. So you have this activating phosphate here. So now it’s able to phosphorylate many other proteins. So now it’s an active kinase. So it also has many substrates that it can phosphorylate. But you’ve also done is to release the SH2 domain and the SH3 domain from their interactions with the kinase core to now interact with other proteins. And so, in particular, if you look at SH2 domain it’s now serving as a docking site for another phosphotyrosine that belongs to another protein. And so in this way, by activating one kinase, and introducing several different phosphotyrosines, you nucleate a molecular complex. And these can be very large and many biological events radiate from that single activation of kinase. That then functions to integrate many other molecules into a biological response. So those are two examples and what I would like to do in the next lecture because these kinases are so important for disease, they have become important structural targets. And so in the next lecture I’d like to talk about the structure of the kinase using PKA as an example but try to help you understand how a kinase works as a molecule. And then after that I’ll talk about how it’s regulated and localized.


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